1. Field of the Invention
The present invention relates to a novel recombinant thermostable enzyme which converts maltose into trehalose.
1. Description of the Prior Art
Trehalose is a disaccharide which consists of 2 glucose molecules linked together with their reducing groups, and, naturally, it is present in bacteria, fungi, algae, insects, etc., in an extremely-small quantity. Having no reducing residue within the molecule, trehalose does not cause an unsatisfactory browning reaction even when heated in the presence of amino acids or the like, and because of this it can advantageously sweeten food products without fear of causing unsatisfactory coloration and deterioration. However, trehalose is far from being readily prepared in a desired amount by conventional methods, and, actually, it is not scarcely used for sweetening food products.
Conventional methods are roughly classified into 2 groups, i.e. the one using cells of microorganisms and the other employing a multi-enzymatic system wherein enzymes are allowed to act on saccharides. The former, as disclosed in Japanese Patent Laid-Open No.154,485/75, is a method which comprises allowing to grow microorganisms such as bacteria and yeasts in a nutrient culture medium, and collecting trehalose from the resultant culture. The latter, as disclosed in Japanese Patent Laid-Open No.216,695/83, is a method which comprises providing maltose as a substrate, allowing a multi-enzymatic system using maltose- and trehalose-phosphorylases to act on maltose, and isolating the formed trehalose from the reaction system. Although the former facilitates the growth of microorganisms without special difficulty, it has a drawback that the resultant culture only contains at most 15 w/w % trehalose, on a dry solid basis (d.s.b.). While the latter enables the separation of trehalose with a relative easiness, but it is theoretically difficult to increase the trehalose yield by allowing enzymes to act on substrates at a considerably-high concentration because the enzymatic reaction per se is an equilibrium reaction of 2 different types of enzymes and the equilibrium point constantly inclines to the side of forming glucose phosphate.
In view of the foregoing, the present inventors energetically screened enzymes which directly convert maltose into trehalose, and have found that microorganisms belonging to those of the genera Pimelobacter and Pseudomonas, as disclosed in Japanese Patent Application No.199,971/93, produce an absolutely novel enzyme which forms trehalose when acts on maltose. This means that trehalose can be prepared from maltose as a material which is readily available in quantity and at low cost, and the use of the enzyme would completely overcome all the aforesaid objects.
It was found that all the enzymes from these microorganisms have an optimum temperature of about 20-40.degree. C. which seems some how insufficient for trehalose production in their thermostability. It is recognized in this field that the saccharification of starch and amylaceous substances should be generally reacted at a temperature of over 55.degree. C.: If the saccharification reaction is effected at a temperature of 55.degree. C. or lower,, bacterial contamination is enhanced to lower the pH of the reaction mixtures and to inactivate enzymes used, followed by remaining a relatively large amount of substrates intact. If the saccharification reaction is effected by using enzymes with poor thermostability, a great care should be taken for the pH changes, and, once a pH lowering occurs, alkalis should be quickly added to the reaction mixtures to increase the pH.
In view of the foregoing, the present inventors further studied on thermostable enzymes with such activity and have found that enzymes, produced from microorganisms of the genus Thermus such as a microorganism of the species Thermus aquaticus (ATCC 33923), effectively convert maltose into trehalose without being substantially inactivated even when reacted at a temperature of over 55.degree. C. These enzymes, however, are not sufficient in enzyme producing activity, and this leads to a problem of that an industrial scale production of trehalose inevitably requires a considerably large scale cultivation of such microorganisms.
Recombinant DNA technology has made a remarkable progress in recent years. At present, even an enzyme, whose total amino acid sequence is not revealed, can be readily prepared in a desired amount, if a gene encoding the enzyme was once isolated and the base sequence was decoded, by preparing a recombinant DNA containing a DNA which encodes the enzyme, introducing the recombinant DNA into microorganisms or cells of plants or animals, and culturing the resultant transformants. Under these circumstances, urgently required are to find a gene encoding the above thermostable enzyme and to decode the base sequence.